The action of EDTA on human alkaline phosphatases

BIOCHIMICAET BIOPHYSICAACTA

363

BBA 65598 T H E ACTION OF E D T A ON HUMAN A L K A L I N E P H O S P H A T A S E S

R. A. J. CONYERS, D. J. BIRKETT, F. C. NEALE, S. POSEN AND JOAN BRUDENELLWOODS Department of Medicine, The University of Sydney, and Department of Biochemistry, Sydney Hospital, Sydney, New South Wales (Australia)

(Received December 23rd, 1966)

SUMMARY

The effects of E D T A on the human alkaline phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.I.3.i ) of bone, intestine and placenta, have been studied b y means of automated methods employing controlled concentration gradients. E D T A has three effects on these phosphatases: (a) In the presence of excess substrate, low concentrations of E D T A (10-5-10 -3 M) cause a loss of phosphatase activity which is the same irrespective of the E D T A concentration. (b) Above 10 -3 M E D T A bone and intestinal phosphatases display an increasing loss in activity with increasing concentrations of EDTA. Placental phosphatase, however, displays a progressive gain in activity with increasing concentration of EDTA. (c) Preincubation of the phosphatases with E D T A results in a time-dependent inactivation which is not reversed b y dilution. This inactivation is also temperature-dependent and p H dependent. Alkaline phosphatases from different tissues show different susceptibilities to the irreversible inactivation b y EDTA. The controlled concentration gradients were also used to study the kinetics of the action of E D T A on these phosphatases. Complex kinetics were observed with all alkaline phosphatases.

INTRODUCTION Alkaline phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) are usually regarded as metalloenzymes 1-4 and, as such s, they have been the subject of several studies6, 7 employing metal-complexing agents. This paper reports the results of our investigations into the effects of E D T A on human phosphatases*. The automated techniques employed, particularly the controlled concentration gradients of inhibitors and substrates have been described in a previous publication 8.

" The term "phosphatase" in this paper refers to alkaline phosphatase unless otherwise indicated. Biochim. Biophys. Acta, 139 (i967) 363-371

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R . A . J . CONYERS et aI.

MATERIALS AND METHODS

Enzymes Macroscopically n o r m a l h u m a n bone a n d i n t e s t i n e were o b t a i n e d at a u t o p s y a n d a h u m a n p l a c e n t a was o b t a i n e d after a n o r m a l confinement. E a c h tissue was homogenised in a W a r i n g blendor for 2 rain w i t h approx. 2 vol. of glass-distilled water. 5 vol. of chloroform w a t e r were a d d e d to the h o m o g e n a t e a n d the m i x t u r e was allowed to a u t o l y s e b y s t a n d i n g at room t e m p e r a t u r e for 3 days. The solid m a t t e r was r e m o v e d b y centrifugation for 20 min at 4 ° (650 × g). 3 vol. of cold e t h a n o l were a d d e d to the s u p e r n a t a n t . This m i x t u r e was then s t i r r e d c o n t i n u o u s l y for 30 rain at - - I O °. F u r t h e r centrifugation at 4 ° for 20 rain (IO 400 × g) p r o d u c e d a p r e c i p i t a t e which was washed w i t h a few ml of cold e t h a n o l a n d recentrifuged. The p r e c i p i t a t e o b t a i n e d was dried over calcium chloride a n d the powder was stored at --lO% Crude e x t r a c t s of p l a c e n t a a n d bone were also employed. These were p r e p a r e d b y homogenising the tissues in o . o i M Tris-HC1 buffer (pH 7.4) at o °. The h o m o g e n a t e was filtered t h r o u g h W h a t m a n No. i p a p e r a n d the filtrate was used w i t h o u t f u r t h e r purification. P l a c e n t a l h o m o g e n a t e was s u b j e c t e d to the procedure of GORDON et al2 b y the C o m m o n w e a l t h Serum L a b o r a t o r i e s (C.S.L.), Melbourne (Australia). The freeze-dried p o w d e r o b t a i n e d b y this procedure which consists largely of a l b u m i n b u t which also cont a i n s p l a c e n t a l alkaline p h o s p h a t a s e was dissolved in Ringer l a c t a t e (5 %, w/v) and this solution was used as an a l t e r n a t i v e p r e p a r a t i o n of p l a c e n t a l alkaline p h o s p h a t a s e . All e n z y m e p r e p a r a t i o n s , before being used, were d i a l y z e d for 24 h in the cold room against 5 changes of 0.05 M Tris-HC1 buffer (pH 7.4).

Assay procedure The buffers, a s s a y procedure a n d a u t o m a t e d techniques have been described b y BIRKETT et al. s a n d KITCHENER et al. 1°. All glassware a n d Technicon A u t o A n a l y z e r t u b i n g were washed in lO -8 M E D T A followed b y n u m e r o u s rinses in glass-distilled water. No metals were a d d e d to the solutions except where indicated.

Assay of enzyme activity in the presence of E D T A The Technicon A u t o A n a l y z e r was used to a s s a y enzyme a c t i v i t y 11 a n d to establish several controlled c o n c e n t r a t i o n g r a d i e n t s s of E D T A which e n t e r e d t h e a s s a y m i x t u r e t o g e t h e r w i t h the s u b s t r a t e solution. The final a s s a y m i x t u r e 1° c o n t a i n e d 6.5" IO -s M d i s o d i u m p h e n y l o r t h o p h o s p h a t e in 0.025 M c a r b o n a t e - b i c a r b o n a t e buffer (pH IO) a n d E D T A in final concentrations v a r y i n g from o to lO -1 M. Results were expressed as K i n g - A r m s t r o n g units TM per IOO ml of enzyme solution. The effect of I , I o - p h e n a n t h r o l i n e was s t u d i e d in a similar manner. The i n c u b a t i o n period was approx. 5 min, a correction factor being a p p l i e d for m i n o r v a r i a t i o n s from this figure. R e a c t i o n velocities were linear over this period a n d at this s u b s t r a t e concentration regardless of w h e t h e r or not inhibitors were present in the a s s a y system.

Pre-incubation with E D T A Various concentrations of E D T A were p r e p a r e d in o . o i M Tris-HC1 buffer (pH 7.4) or in 0.025 M c a r b o n a t e - b i c a r b o n a t e buffer (pH IO). These E D T A - b u f f e r solutions were placed in a w a t e r - b a t h at 37 °, enzyme was a d d e d to give an original a c t i v i t y of

Biochim. Biophys. Acla, 139 (1967) 363-371

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15-3o King-Armstrong units per ioo ml, aspiration of the mixture was commenced immediately and continued for 30 min. The method of continuous monitoring of enzyme activity has been previously described8, TM. In this set of experiments the enzyme-EDTA solution was subject to a I6-fold dilution in the AutoAnalyzer manifold so that the highest concentration of EDTA in the pre-ineubation mixture (lO -3 M) resulted in an EDTA concentration of 6.25" lO -5 M during incubation. Magnesium sulphate or sodium fl-glycerophosphate was added to the preincubation mixture in some of the experiments so as to give a concentration of lO-4 M in the pre-incubation mixture. The volume changes caused by the addition of these substances never exceeded 1%. Determination of Michaelis constants This was performed in o.o5 M Tris-HC1 (pH 9) by the automated method described by BIRKETT et al. 8 at an incubation time of 45 sec. K ~ and Vmaxwere calculated according to the method of LINEWEAVERAND BURK14 on an English Electric K D F 9 computer by means of an Algol program written by Mr. M. MANTON. The substrate gradient was o-o.15 mM disodium phenylorthophosphate and the enzyme activities used were in the range lO-15 King-Armstrong units per IOO ml when assayed under optimal conditions 1°.

RESULTS E n z y m e inhibition as a function of E D T A concentration Fig. I shows the effect of different concentrations of EDTA in the assay system. In the presence of substrate and lO -5 M EDTA these phosphatases exhibited a loss of activity which varied according to the source of enzyme. Bone phosphatase, in 4 experiments, lost a mean of 18.5% of its original activity (range 17-21°/o ) while placental phosphatase (7 experiments) lost a mean of 15.3% of its activity (range 13-18%). Intestinal phosphatase in 5 experiments lost a mean of only 6.6% of its original activity (range 4-1o%). When the concentration of EDTA was increased from IO 5 M to IO 3 M EDTA there was no further loss of activity by any of the phosphatase preparations. When the concentration of EDTA in the assay system exceeded 3" lO-8 M, both intestinal and bone phosphatase exhibited a progressive diminution in activity with increasing concentrations of EDTA. This loss was not affected by changes in incubation time within the range I-IO rain. At lO -1 M EDTA, intestinal phosphatase showed 45 % of its original activity while only 30% of the original activity of bone phosphatase remained. Placental phosphatase, however, over the range lO -3 M - I o -1 M EDTA displayed a progressive gain in activity so that at 2" IO-~ M EDTA the mean activity was 122 °/o of the original. Further increases in EDTA concentrations up to 1.9. io 1 M did not lead to a further increase in placental alkaline phosphatase activity. A similar pattern of activation was obtained in o.oi M Tris-HC1 buffer at pH 9.0. But at pH 8.0 there was only negligible activation. The activation of placental phosphatase which occurred in the presence of 6. 5 mM phenylorthophosphate was not seen at a sulzstrate concentration of o.15 mM. These effects of EDTA were uninfluenced by the mode of preparation of the various phosphatases. Biochim. Biophys. Ac/a, 139 (1967) 363 37~

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Fig. I. The change in activity of three h u m a n alkaline p h o s p h a t a s e s as a function of the concent r a t i o n of E D T A in the assay mixture. The E D T A concentrations were varied b y m e a n s of controlled concentration gradients s and the composite curve s h o w n for each enzyme is the result of 4 such gradients over 4 concentration ranges (IO-S-IO-4 M, lO-4-1o -3 M, lO-3-1o -2 M, and lO-2-1o -1 M). The s u b s t r a t e was disodium p h e n y l o r t h o p h o s p h a t e (6. 5. lO -3 M) in o.o25 M c a r b o n a t e ~ i c a r b o n a t e buffer (pH io) and the incubation t e m p e r a t u r e was 37 °. lOO% is the activity of each enzyme preparation under these conditions in the absence of added EDTA. Note the large n u m b e r of experimental points obtainable by this method.

Pre-incubation with E D T A Fig. 2 shows the effect of pre-incubation with various concentrations of EDTA on the activity of purified bone, intestinal and placental alkaline phosphatases. In Tris-HC1 buffer (pH 7.4) EDTA caused a time-dependent, temperature-dependent exponential inactivation which varied with the concentration of EDTA and according to the tissue of origin of the enzyme, and which was not reversed by dilution. In the presence of lO -3 M EDTA, at 37 ° the mean half life of placental phosphatase was 6 min (3 experiments, range 5-7 rain), that of bone phosphatase 2 min (4 experiments, range 0.5-3 min) and that of intestinal phosphatase less than I rain (3 experiments). These times were not affected by an increase in the buffer strength from o.oi M to 0.05 M, or by the degree of purification of the enzymes, although C.S.L. placental alkaline phosphatase was more labile than the other preparations of placental phosphatase. When the pH of the pre-incubation mixture was increased to io.o the inhibitory effect of EDTA was reduced. In 1o -8 M EDTA the half life of placental phosphatase was 38 min, that of bone phosphatase 3.5 rain, while intestinal phosphatase had a half life of 6.5 rain. Addition of substrate and magnesium to the pre-incubation mixture Fig. 3 shows tile effect of magnesium sulphate and/5-glycerophosphate on the activity of C.S.L. placental phosphatase incubated in lO-4 M EDTA at pH 7.4. In the absence of EDTA or other added reagents, C.S.L. placental phosphatase showed a loss of activity of approx. 5% over 30 min at 37 °. In the presence of lO-4 M magnesium, no loss of activity occurred 15-17. However, the presence of lO -4 M/~-glyceroBiochim. Biophys. Acta, 139 (1967) 363-371

EDTA AND ALKALINE PHOSPHATASES

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phosphate did not protect this preparation against the 5 % loss of activity over 3o min at this temperature. The presence of magnesium in the pre-incubation mixture prior to the addition of enzyme, afforded complete protection against EDTA inactivation at pH 7.4. IL instead, the magnesium was added to the pre-incubation mixture 5 min (or IO rain) Minutes,

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Fig. 2. Decrease in activity of purified h u m a n alkaline p h o s p h a t a s e s as a function of the E D T A concentration and the duration of pre-incubation with E D T A at 37 °. The pre-incubation mixtures contained E D T A at the concentrations indicated , in o.oi M Tris-HC1 (pH 7.4). These were diluted b y a factor of 16 before assay. E n z y m e activity was monitored continuously 18.

after the addition of enzyme, there was an immediate, though not complete, return of activity (from 60% to 86% at 5 rain and from 41% to 59% at IO rain). The activity to which the enzyme had been restored then remained constant throughout the period of observation. A similar protective effect of magnesium in the pre-incubation mixture was observed at pH IO. At this pH, at 37 °, andin io-SM EDTA, the half lives of bone, intestinal and placental phosphatases were increased by magnesium to 30 min, 40 rain and no loss at all (over 30 min), respectively. When fl-glycerophosphate was added to the pre-incubation mixture before Biochim. Biophys. Acta, 139 (1967) 363-371

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Fig. 3. The effect of m a g n e s i u m sulphate and fl-glycerophosphate on the activity of h u m a n placental alkaline p h o s p h a t a s e incubated with lO -4 M E D T A . E x p e r i m e n t a l procedure was the same as outlined in Fig. 2. Final molarities of Mg ~+ and fl-glycerophosphate in the pre-incubation m i x t u r e were each lO -4 M. E n z y m e was added at zero time and the arrows indicate the times at which further additions were made. x , E D T A only; O, E D T A + Mg2+; m, E D T A , Mg 2+ added at 5 min; A, E D T A , Mg 2+ added at io min; C), E D T A + fl-glycerophosphate; [[], E D T A , flglycerophosphate added at 5 min.

placental phosphatase (C.S.L.), the rate of decay of enzyme activity was reduced considerably. In a pre-incubation mixture containing IO 4 M EDTA the half life of this enzyme preparation was 8 min. In tile presence of fl-glycerophosphate this was converted to a half life of 135 min. The addition of fl-glycerophosphate to the preincubation mixture 5 rain after the addition of enzyme also reduced the rate of decay of enzyme activity. Unlike magnesium, fl-glycerophosphate caused only a 5 % return in enzyme activity. The addition to the pre-incubation mixture at 5 rain of magnesium and fl-glycerophosphate together, produced results very similar to the effects of magnesium alone.

Effects of p henanthroline I,Io-Phenanthroline in concentrations up to lO -3 M had no effect on placental Biochim. Biophys. Acta, 139 (1967) 363-371

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phosphatase in the presence of substrate and at pH IO.O. Pre-incubation of placental phosphatase with lO -4 M and lO -3 M I,Io-phenanthroline at pH 7-4 was also without effect.

Lineweaver-Burk plots Fig. 4 shows double reciprocal plots computed from data obtained by the automated method of BIRKETT et al. s. Forty five substrate concentrations were used to calculate linear regressions for each EDTA concentration. 5" lO-5 M EDTA reduced both Vmax. and K ~ of placental phosphatase. As the concentration of E D T A was

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Fig. 4. L i n e w e a v e r - B u r k plots of the effect of E D T A on h u m a n placental alkaline phosphatase. The assay m i x t u r e contained E D T A in concentrations as indicated and p h e n y l o r t h o p h o s p h a t e in o.o 5 M Tris-HC1 buffer, p H 9,o at 37 °. Metal ions were not added. The Technicon AutoAnalyzer was used for the assay procedure and to establish a controlled concentration gradient of o - o . i 5 mM p h e n y l o r t h o p h o s p h a t e in the assay mixture. E n z y m e activities at 45 s u b s t r a t e concentrations were used for each E D T A concentration to c o m p u t e the linear regressions shown. I n the absence of E D T A the Km of placental p h o s p h a t a s e was o.o33 mM.

increased, both Vmax and Km increased, until at i . lO -2 M the Vmax was in excess of the initial value. Further increases in EDTA concentration produced only increases in K,~ without any concomitant increase in Vmax. DIXON plots is for placental phosphatase were obtained both by replotting the data in Fig. 4 and by using controlled concentration gradients of EDTA at various substrate concentrations. As would be expected from Fig. 4 curved plots were obtained at all substrate concentrations. DISCUSSION

These results indicate that EDTA has three effects on human alkaline phosphatases. The first, occurring with low concentrations of EDTA in the assay system, is an instantaneous inhibition, apparently independent of variations in the EDTA concentrations within the range i o - S - i o -8 M. At concentrations of EDTA above lO -3 M in the assay system, the second effect is seen. Bone and intestinal phosphatase display an increasing loss in activity with increasing EDTA concentrations, while placental Biochim. Biophys. Acta, 139 (1967) 363-371

37 °

R.A.J. CONYERS et al.

phosphatase shows a progressive gain in activity. Pre-incubation of the phosphatases with EDTA produces the third effect: a time-dependent inactivation which is not reversed by dilution. It is suggested that the first effect is due to some activator, possibly a metal 19-21, which is contaminating the enzyme preparations or tile commercial reagents employed 22-24. Such a postulate would explain the reduction in Vmax of placental and bone phosphatase (Fig. i) upon the addition of small quantities of E D T A and the absence of any further reduction in spite of a subsequent increase in the E D T A concentration of the assay system. The apparently insignificant effect of small concentra tions of EDTA on intestinal phosphatase is attributed to structural differences between this enzyme and other alkaline phosphatases 25-27. The second effect of EDTA on bone and intestinal alkaline phosphatases commences at EDTA concentrations above 10 -3 M (Fig. I). It m a y be due to the chelation of some divalent cation necessary for enzyme activity1, 2s 30, or to a direct effect of EDTA on the enzyme molecule v. Placental phosphatase differs from tile other phosphatases by being activated b y EDTA, a property which it shares with various non-phosphatase enzymes 22,31-33.A review of the mechanisms whereby these enzymes m a y be activated by EDTA 22-24,31--40 is beyond the scope of this paper. In the case of phosphatases, tile activating effect m a y be confined to the placental enzyme because of its very stable protein structure. Placental phosphatase differs from other human phosphatases in its amino acid composition 41 and in its stability to heat TM,citrate 10 and urea s. Since tile phosphatases used in this work were crude preparations the complex kinetics shown in Fig. 4 will not be discussed in this paper. The third effect of E D T A ~ i . e . the time-dependent inactivation which is not reversed by dilution (Fig. 2)--is seen with all three human phosphatases and has previously been described with other mammalian phosphatases e,21,3°. This third effect is readily explained by the postulate that EDTA removes a functionally essential metal (probably zinc)6,21,3° though it is not possible to deduce from this work whether EDTA binds the metal in situ and slowly induces conformational changes in the protein structure, or whether the E D T A immediately removes the metal from the protein, leaving an unstable enzyme molecule which then undergoes denaturation 42,43. AGUS, Cox AND GRIFFIN4~ concluded that E D T A probably removes the metal from the metalloenzymes. The different rates of inactivation displayed by the various enzymes during preincubation with EDTA are attributed to structural differences between the various phosphatasesS,le, 25-27. Time-dependent activation by pre-incubation with EDTA, described by MILSTEIN for phosphoglucomutase 31, was not observed in these experiments. ACKNOWLEDGEMENTS We gratefully acknowledge the interest of Dr. J. DONE, Department of Biochemistry, University of Sydney. Miss C. ROTHERO, Mr. R. MONEY, and Mr. J. COLLINS are hereby thanked for their assistance. This work was supported b y the N.S.W. State Cancer Council and the Edanros Research Foundation, Inc., New York. Biochim. Biophys. Acta, 139 (1967) 363-371

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Biochim. Biophys. Acta, 139 (1967) 363-371